Abstract Morbidity and mortality associated with Chagas disease in the American continent exceed better-known conditions such as malaria, tuberculosis, or AIDS. Millions of people are affected by this trypanosomiasis. No vaccines are available to prevent this disease and drug treatments have serious side effects and are not completely effective. The study of metabolic pathways in these parasites that may be essential for their survival but may not find an equivalent counterpart in their host could lead to the development of specific inhibitors as possible means of controlling the parasites without damaging the hosts. A drawback for these studies in Trypanosoma cruzi, the etiologic agent of Chagas disease, has been the lack of genetic tools to demonstrate the essentiality of these metabolic pathways. Few genetic tools were available to work with T. cruzi until our recent introduction of the CRISPR/Cas9 technique for gene knockout, gene complementation, and endogenous gene tagging. However, key obstacles to defining targets that can be used for chemotherapy have been the lack of ability to reversibly and temporally regulate gene expression in these parasites, in comparison to what is possible in the related parasite Trypanosoma brucei. The fastest method for the generation of conditional mutants in T. brucei is the use of RNA interference. This pathway is absent in T. cruzi. Complementation with a tetracycline-regulated extra copy of the gene of interest to allow the knockout of the endogenous alleles in a cell line stably expressing a tet repressor and the T7 RNA polymerase (inducible knockout) has also been successfully employed in T. brucei. However, attempts to develop similar methods in T. cruzi have failed until now or the methods have not been reproduced in other laboratories. In this exploratory project, we propose the test of alternative methods to conditionally regulate gene expression at the translational or posttranslational levels. With our recent success in establishing a CRISPR/Cas9 system for T. cruzi endogenous C-terminal tagging we can now test these approaches with genes encoding proteins that have been proposed as potential drug targets. We propose to use two methods for inducible downregulation of genes in T. cruzi in combination with CRISPR/Cas9. One method for the inducible loss of gene function mediated at the mRNA level is the use of riboswitches, which are naturally occurring self-cleaving RNAs (ribozymes) that can be modified to respond to ligands. Another method successfully applied to other unicellular eukaryotes is the auxin-inducible degron system in which the plant hormone auxin directly induces rapid degradation of the protein of interest by targeting it to the proteasome. Our hypothesis is that these methods could be used in combination with CRISPR/Cas9 for inducible downregulation of genes in T. cruzi.